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1.
Inflamm Res ; 55(9): 393-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-17122961

ABSTRACT

METHODS: The effect of histamine on inositol phosphate generation and interleukin-6 (IL-6) release from the synovial sarcoma cell line SW982 was investigated. RESULTS: SW982 cells express functional H1 and H2 receptors. The H1 receptor antagonist [3H]-mepyramine binds to membranes from SW982 cells with high affinity and the binding was potently blocked by H1 antagonists. Histamine potently stimulated phosphoinositide (PI) hydrolysis and Ca2+ mobilization with EC50 of 4.0 +/- 0.8 microM and 1.3 +/- 0.6 microM respectively and these activities were blocked by the H1 selective antagonist mepyramine. Histamine (EC50 = 1.8 +/- 1.1 microM) stimulated the release of IL-6 that was attenuated by selective H1 antagonists. The PKC inhibitor, GF1090203X, blocked the histamine stimulated IL-6 release. The H2 selective antagonist, cimetidine, had no significant effect on histamine-induced PI turnover, Ca2+ mobilization and IL-6 release. CONCLUSION: We conclude that histamine stimulates IL-6 release from SW982 cells by binding to the H1 receptor and this is coupled to the PI/PKC signal transduction pathway. Development of an H1 antagonist that inhibits the release of IL-6 from synoviocytes may be beneficial for the treatment of inflammatory joint disease.


Subject(s)
Histamine/pharmacology , Interleukin-6/metabolism , Receptors, Histamine H1/drug effects , Cell Line, Tumor , Cimetidine/pharmacology , Histamine H1 Antagonists/pharmacology , Histamine H2 Antagonists/metabolism , Histamine H2 Antagonists/pharmacology , Humans , Indoles/pharmacology , Maleimides/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Histamine H1/metabolism , Sarcoma, Synovial
2.
J Clin Invest ; 107(2): 173-80, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11160133

ABSTRACT

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Insulin/pharmacology , Peptide Fragments/pharmacology , Prediabetic State/immunology , T-Lymphocytes/drug effects , Adolescent , Adult , Cells, Cultured , Diabetes Mellitus, Type 1/blood , Female , Humans , Immunodominant Epitopes/pharmacology , Interferon-gamma/immunology , Lymphocyte Activation/drug effects , Male , Prediabetic State/blood , Risk Factors , T-Lymphocytes/immunology
3.
Ann Neurol ; 48(5): 758-65, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11079539

ABSTRACT

We assessed the immune response induced in multiple sclerosis (MS) patients who had received NBI-5788, an altered peptide ligand (APL) designed from an immunodominant region (83-99) of the neuroantigen myelin basic protein (MBP) (5, 10, or 20 mg subcutaneously weekly for 4 weeks). The mean frequency of NBI-5788-responsive T cells (stimulation index > 3) in MS patients treated with the APL was 35.8 +/- 12.8% (n = 7) compared with a mean frequency of 6.2 +/- 1.3% (n = 7) for the untreated patients. The mean frequency of whole MBP-responsive T cells in MS patients treated with the APL was not significantly different from that of untreated patients (16.4 +/- 5.7% vs 18.0 +/- 6.3%, respectively). NBI-5788-reactive T-cell lines generated from NBI-5788-treated patients exhibited an increased frequency of cross-reactivity with MBP peptide 83-99 compared with NBI-5788-reactive lines from control MS patients. Cytokine secretion by APL-reactive T-cell lines from NBI-5788-treated MS patients was more frequently T-helper 2-like compared with T-cell lines from untreated MS patients. These results demonstrate that subcutaneous administration of a soluble APL based on the MBP peptide 83-99 in MS patients can induce an APL-reactive immune response in which T lymphocytes cross-reactive with the immunodominant neuroantigen MBP secrete anti-inflammatory cytokines. The significant heterogeneity in immune response between individuals indicates the need for clinical laboratory correlation during the course of therapeutic trials.


Subject(s)
Cytokines/immunology , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Myelin Basic Protein/administration & dosage , Peptide Fragments/administration & dosage , Peptide Fragments/pharmacology , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/immunology , Adult , Antibody Specificity/immunology , Cytokines/drug effects , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Multiple Sclerosis/pathology , Myelin Basic Protein/drug effects , Myelin Basic Protein/immunology
4.
Gene ; 238(2): 471-8, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10570975

ABSTRACT

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.


Subject(s)
Immediate-Early Proteins , Intercellular Signaling Peptides and Proteins , Kidney Neoplasms/genetics , Oncogene Proteins, Viral/genetics , Proto-Oncogene Proteins/genetics , Tyrosine/metabolism , Wilms Tumor/genetics , 3T3 Cells , Amino Acid Sequence , Animals , Cell Division , Cloning, Molecular , Connective Tissue Growth Factor , DNA, Complementary , Dexamethasone/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Molecular Sequence Data , Nephroblastoma Overexpressed Protein , Oncogene Proteins, Viral/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tumor Cells, Cultured
5.
J Immunol ; 163(3): 1611-8, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10415066

ABSTRACT

Eotaxin is a potent eosinophil chemoattractant that plays an important role in regulating eosinophil tissue levels both in healthy individuals and in diseases associated with significant eosinophil infiltrates, such as the allergic inflammation observed in asthma. Here, we demonstrate that treatment of eosinophils with eotaxin induces the phosphorylation of the mitogen-activated protein kinases (MAPKs) p42 and p44, leading to kinase activation. Blockade of MAPK activation by the MAPK kinase inhibitor PD98059 leads to a dramatic decrease in eotaxin-induced eosinophil rolling in vivo and chemotaxis in vitro. This blockade in the leukocyte migration process is consistent with the observed inhibition of actin polymerization and rearrangement within the eosinophil following treatment with MAPK inhibitor. It is suggested, therefore, that the intrinsic mechanism of eotaxin-induced eosinophil rolling and migration involves activation of the p42/p44 MAPK, possibly through regulation of the cytoskeletal rearrangements necessary for chemotaxis.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Chemokines, CC , Chemotaxis, Leukocyte/physiology , Cytokines/pharmacology , Eosinophils/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases , Actins/antagonists & inhibitors , Actins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Migration Inhibition , Chemokine CCL11 , Chemotaxis, Leukocyte/drug effects , Dose-Response Relationship, Immunologic , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Eosinophils/drug effects , Eosinophils/metabolism , Flavonoids/pharmacology , Humans , Immunophenotyping , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Time Factors
6.
Hum Immunol ; 59(11): 679-89, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9796736

ABSTRACT

Changes in peptide antigen concentration or structure can have a profound effect on T cell responsiveness by inducing selected T cell effector functions. In this study, we have compared the biological responses of an MBP83-99-specific human Th0 T cell clone (TCC) stimulated with increasing concentrations of native peptide or an altered peptide ligand (APL). Our results show that the hierarchy of response thresholds for proliferation and cytokine secretion is similar for native peptide and APL. However, because a much higher concentration of the APL is required to evoke the same degree of response, the cytokine profile is shifted towards a Th2-like response relative to the same concentration of native peptide. In addition, we observed qualitative differences in TCR signal transduction triggered by native peptide and a weak agonist APL even at concentrations that elicit similar biological responses. Thus, the relationship between TCR signaling and biological responses may be more complex than previously recognized.


Subject(s)
Adaptor Proteins, Signal Transducing , Cytokines/biosynthesis , Membrane Proteins/immunology , Myelin Basic Protein/immunology , Peptide Fragments/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Amino Acid Substitution , B-Lymphocytes , Binding Sites , Cell Division , Cell Line , Coculture Techniques , GRB2 Adaptor Protein , HLA-DR Antigens/metabolism , HLA-DRB1 Chains , Humans , Immunodominant Epitopes , Membrane Proteins/metabolism , Molecular Sequence Data , Myelin Basic Protein/chemistry , Peptide Fragments/chemistry , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Helper-Inducer/cytology , ZAP-70 Protein-Tyrosine Kinase , src-Family Kinases/metabolism
7.
Eur J Immunol ; 28(6): 1894-901, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9645371

ABSTRACT

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83-99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83-99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83-99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81-95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83-99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.


Subject(s)
Epitopes, T-Lymphocyte/immunology , Immunodominant Epitopes/immunology , Multiple Sclerosis/immunology , Myelin Basic Protein/immunology , Peptide Fragments/immunology , T-Lymphocytes/immunology , Alanine/immunology , Cells, Cultured , Cross Reactions , Epitopes, T-Lymphocyte/chemistry , Herpesvirus 2, Human/immunology , Humans , Immunodominant Epitopes/chemistry , Interferon-gamma/analysis , Interleukin-10/analysis , Lysine/immunology , Myelin Basic Protein/chemistry , Papillomaviridae/immunology , Peptide Fragments/chemistry , Peptides/chemistry , Peptides/immunology , Phenylalanine/immunology , Receptors, Antigen, T-Cell/immunology , Structure-Activity Relationship , Viral Proteins/immunology
8.
J Neuroimmunol ; 74(1-2): 149-58, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9119968

ABSTRACT

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/physiopathology , Myelin Basic Protein/pharmacology , Peptide Fragments/pharmacology , Animals , Cell Death , Cytokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Ligands , Lymphocyte Activation , Mice , Mice, Inbred Strains , Myelin Basic Protein/immunology , Peptide Fragments/immunology , Recurrence , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/physiology
9.
Cell Immunol ; 176(2): 103-12, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9073382

ABSTRACT

Stress elicits a wide range of physiological changes involving the nervous, endocrine, and immune systems. Corticotropin-releasing factor (CRF) plays a key role in orchestrating this response, activating both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, resulting in release of corticosteroids. The present study examines the immunological phenotype and responsiveness of CRF-transgenic (CRF-Tg) mice. The immune system of the CRF-Tg animals has profound changes compared to littermate controls, including a marked reduction in both cell number and immune responsiveness. There were also phenotypic changes in the lymphocytic composition of the various lymphoid organs, most notably in the spleen, where CRF-Tg mice had a greater percentage of T lymphocytes compared to littermate controls. Adrenalectomy of CRF-Tg reversed the immunological phenotype observed and restored immune responsiveness. These results demonstrate that CRF overexpression leads to profound impairment on lymphocyte development and function mediated via corticosteroids.


Subject(s)
Adrenalectomy , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/immunology , Immunosuppressive Agents/immunology , Animals , Corticotropin-Releasing Hormone/antagonists & inhibitors , Female , Immunophenotyping , Immunosuppressive Agents/antagonists & inhibitors , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic
10.
J Neuroimmunol ; 70(2): 113-22, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8898719

ABSTRACT

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL 1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL 1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [125I]-recombinant human IL 1 alpha or [125I]-recombinant human IL 1 beta. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.


Subject(s)
Proteins/genetics , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Binding, Competitive , Cloning, Molecular , DNA, Complementary/genetics , Humans , In Situ Hybridization , Interleukin-1/metabolism , Interleukin-18 Receptor alpha Subunit , Ligands , Membrane Proteins/genetics , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , Rats , Receptors, Cell Surface/metabolism , Receptors, Interleukin , Receptors, Interleukin-1/chemistry , Receptors, Interleukin-18 , Sequence Alignment , Sequence Homology, Amino Acid , Tissue Distribution
11.
J Pharmacol Exp Ther ; 278(2): 455-61, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8768691

ABSTRACT

The regulation of brain corticotropin-releasing factor (CRF)-binding protein (BP), an endogenous modulator of the CRF family of neuropeptides, has been difficult to pursue due to a lack of basal expression in a known cell line or primary cells in vitro. In light of the ability of intracellular factors to modulate neuronal and glial function, we examined the effects of a variety of signal transduction modulators on CRF-BP expression in cultured astrocytes. In particular, the effect of agents that stimulate protein kinase A and protein kinase C pathways was evaluated. CRF-BP was measured using a ligand immunoradiometric assay. Forskolin, dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine treatment resulted in a dose-dependent increase in CRF-BP levels detected in the medium from astrocytes and neurons. The increase in CRF-BP expression was not due to increased cell proliferation as measured by [3H]thymidine incorporation. In addition, treatment of the astrocytes with phorbol myristate acetate, a protein kinase C activator, caused a robust increase in CRF-BP levels in the medium. Steroids such as dexamethasone, corticosterone, hydrocortisone and, to a lesser extent, dehydroepiandosterone inhibited the stimulated release of CRF-BP from astrocytes. These data define a primary role for intracellular messengers in regulating CRF-BP expression in neurons and astrocytes.


Subject(s)
Astrocytes/drug effects , Carrier Proteins/metabolism , Corticotropin-Releasing Hormone/metabolism , Animals , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/pharmacology , Rats
12.
J Exp Med ; 181(3): 1205-10, 1995 Mar 01.
Article in English | MEDLINE | ID: mdl-7869036

ABSTRACT

TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.


Subject(s)
Dipeptides/pharmacology , Hydroxamic Acids/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Humans , Lymphocyte Activation , Metalloendopeptidases/physiology , Mice , Receptors, Tumor Necrosis Factor/metabolism , Transfection , Tumor Necrosis Factor-alpha/metabolism
14.
Science ; 264(5159): 707-10, 1994 Apr 29.
Article in English | MEDLINE | ID: mdl-8171323

ABSTRACT

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.


Subject(s)
Lymphotoxin-alpha/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Cysteine/chemistry , Humans , Hybridomas , Ligands , Lymphotoxin beta Receptor , Molecular Sequence Data , Receptors, Tumor Necrosis Factor/chemistry , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/immunology , Tetradecanoylphorbol Acetate/pharmacology
15.
J Immunol Methods ; 168(1): 79-89, 1994 Jan 12.
Article in English | MEDLINE | ID: mdl-8288897

ABSTRACT

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.


Subject(s)
Lymphotoxin-alpha/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Animals , Base Sequence , Biological Assay , Cells, Cultured , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Insecta , Lymphotoxin-alpha/genetics , Lymphotoxin-alpha/metabolism , Molecular Sequence Data , Neutralization Tests , Nucleopolyhedroviruses/genetics , Receptors, Tumor Necrosis Factor/genetics , Recombinant Fusion Proteins/biosynthesis , Solubility
16.
J Immunol ; 151(12): 6882-90, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8258697

ABSTRACT

The 80-kDa TNFR (TNFR80) expressed by activated human T cells is constitutively phosphorylated and undergoes limited proteolytic cleavage (shedding) at the cell surface releasing a 40-kDa soluble TNF-binding protein. Triggering of activated T cells through the TCR rapidly increased the rate of TNFR80 shedding > 20-fold more than nonstimulated cells, demonstrating that shedding is a specific, inducible process. The protein kinase inhibitor staurosporine inhibited constitutive phosphorylation and blocked inducible shedding of TNFR80, suggesting that phosphorylation may be important for cleavage of the extracellular domain. However, a deletion mutation of the entire cytoplasmic domain of human TNFR80 was shed when expressed in murine L929 cells, albeit relatively poorly compared with full length receptor. This demonstrates that the cytoplasmic domain is important but not essential for cleavage of the extracellular domain of TNFR80. Moreover, a requirement for phosphorylation of proteins other than the receptor was revealed by the finding that staurosporine completely blocked cleavage of the cytoplasmic deletion mutant. Collectively, these results demonstrate that protein phosphorylation is essential and the cytoplasmic domain is important for regulating the inducible production of soluble TNF-binding proteins by activated effector T cells.


Subject(s)
Receptors, Tumor Necrosis Factor/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Alkaloids/pharmacology , Animals , Base Sequence , Cell Line , Cytoplasm/metabolism , DNA, Complementary/genetics , Gene Expression , Humans , In Vitro Techniques , Kinetics , Lymphocyte Activation , Mice , Molecular Sequence Data , Molecular Weight , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Staurosporine
17.
J Immunol ; 149(12): 3881-8, 1992 Dec 15.
Article in English | MEDLINE | ID: mdl-1281193

ABSTRACT

The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.


Subject(s)
Lymphocyte Activation/immunology , Lymphocytes/metabolism , Lymphotoxin-alpha/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Antigens, CD/immunology , Antigens, CD20 , Antigens, Differentiation, B-Lymphocyte/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , B-Lymphocytes/metabolism , CD56 Antigen , Cell Line , Gene Expression Regulation , Glycoproteins/immunology , Humans , Immunity, Cellular , Interleukin-2/immunology , Killer Cells, Natural/metabolism , Pokeweed Mitogens , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate , Time Factors
18.
J Immunol ; 147(12): 4229-38, 1991 Dec 15.
Article in English | MEDLINE | ID: mdl-1661312

ABSTRACT

The expression of TNF-alpha receptors (TNFR) was examined on a CD4+ T cell hybridoma, transformed T cell lines, CTL clones, and activated T cells from peripheral blood to determine the basis of the immunomodulatory activity of TNF on T cell function. Analyses by ligand cross-linking and competitive binding assays with mAb to the 80-kDa receptor (TNFR-I), demonstrated that the TNFR-I was the predominant receptor expressed on activated CD4+ and CD8+ T cell subsets. However, on T cell leukemic lines, a second, non-TNFR-I binding site was identified, most likely the 55-kDa form (TNFR-II). Additional subsets of T cells were readily distinguished by their expression of TNFR-I and related members of the TNFR gene family (CD40 and CD27). Expression of the TNFR-I was dependent upon the state of T cell activation. Signaling through the TCR for Ag or IL-2R was sufficient to induce TNFR mRNA and protein expression in resting T cells. Multiple sizes of TNFR-I transcripts were detected during T cell activation; however, biosynthetic studies showed these multiple species encode a single protein of 80 kDa. These results, combined with the known ability of TNF to induce IL-2R expression, indicate that TNF and IL-2 form a reciprocating receptor amplification circuit. In contrast, differentiated effector T cells triggered through the TCR or protein kinase C initiated a rapid down-regulation (transmodulation) of the TNFR-I that preceded TNF or lymphotoxin secretion. The mechanism of transmodulation involved proteolytic processing of the mature 80-kDa receptor releasing a soluble 40-kDa fragment. This indicates that a TNF autocrine loop is not likely to form during the response of an effector T cell. Collectively, these results suggest that transcriptional and post-translational modification of the TNFR-I are important control points regulating the expression of this receptor during T cell activation.


Subject(s)
Lymphocyte Activation , Receptors, Cell Surface/analysis , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Animals , Antibodies, Monoclonal/immunology , Cells, Cultured , Precipitin Tests , Rats , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Receptors, Tumor Necrosis Factor , T-Lymphocytes/chemistry
19.
Dig Dis Sci ; 36(9): 1313-9, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1893818

ABSTRACT

The adenohypophyseal polypeptide hormone prolactin is a potent liver mitogen, stimulating cell cycle progression, an effect that appears coupled to increasing protein kinase C activity in membrane and nuclear fractions. Here, we examine whether hepatocyte proliferation, stimulated by partial hepatectomy, is associated with altered serum prolactin or protein kinase C activation. Within 5-15 min of liver resection, serum prolactin concentrations elevate significantly. Protein kinase C activity in hepatic cytosol decreases significantly, and membrane and nuclear PKC activity increase by 30 min. Hypophysectomy prior to partial hepatectomy abrogates any effect of liver resection on protein kinase C activation in the hepatic remnant. Based upon these data, it is suggested that the rapid increase in serum prolactin seen after partial hepatectomy may be linked to protein kinase C activation, which in turn stimulates the hepatic proliferative response that is essential for hepatic regeneration.


Subject(s)
Hepatectomy , Liver Regeneration/physiology , Liver/enzymology , Prolactin/physiology , Protein Kinase C/metabolism , Animals , Cell Division/physiology , Hypophysectomy , Liver/cytology , Male , Pituitary Gland, Anterior/physiology , Prolactin/blood , Rats , Rats, Inbred Strains , Time Factors
20.
Mol Cell Endocrinol ; 79(1-3): 29-35, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1718797

ABSTRACT

We have examined the effect of prolactin (PRL) on growth-related gene expression, protein kinase C (PKC) activity and diacylglycerol (DAG) mass in rat liver. Hepatic levels of messenger (m)RNA for c-myc, ornithine decarboxylase (ODC) and beta-actin increased in a dose-dependent manner within 1 h after PRL administration. Prolactin also caused a transient elevation of liver DAG levels and particulate-associated PKC activity. The PRL-provoked increases in DAG mass and particulate PKC activity were coincident and maximal at 20 min and began declining toward control levels by 30 min. These results suggest a temporal relationship between PRL-stimulated DAG accumulation and PKC activation. Furthermore, the subsequent rapid induction of growth-related gene expression provides new information on the role of PRL as a hepatic mitogen.


Subject(s)
Diglycerides/metabolism , Liver/physiology , Prolactin/pharmacology , Protein Kinase C/metabolism , RNA, Messenger/genetics , Actins/genetics , Aging , Animals , DNA Probes , Enzyme Activation , Gene Expression/drug effects , Genes, myc/drug effects , Histones/genetics , Kinetics , Liver/drug effects , Liver/growth & development , Male , Ornithine Decarboxylase/genetics , RNA/genetics , RNA/isolation & purification , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains
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